On antigenic stimulation, rat basophil leukemic (RBL-243) cells respond with rapid increases in free cytosol calcium concentration, hydrolysis of PI (phosphatidylinositol and its phosphorylated derivatives), and histamine release. These processes depend on the presence of ATP. In the present studies, complete depletion of cellular ATP in 2H3 cells could be achieved only when the cells were treated with antimycin A (0.1 MuM) in a glucose-free medium. Under these conditions, cellular ATP content was less than 1% of the normal level (0.05 plus/minus 0.05 as compared to 5.8 plus/minus 0.06 ug/million in untreated cells) and the release of inositol phosphates (i.e., PI breakdown) and of histamine from the cells were 7% and 13% of the maximum, respectively. The suppression of PI breakdown and histamine release by antimycin A was dose-dependent and were highly correlated (correlation coefficient = P less than 0.001). The ATP-dependent nature of the two processes suggest that a critical phosphorylation step is required for the initiation of PI breakdown and consequently histamine release.